JP6410335B2 - Transformant and production method thereof - Google Patents

Description

  The present invention relates to a transformant and a production method thereof, and more specifically to a transformant (variety) of a eukaryote such as a plant having an increased growth amount (biomass) and a production method thereof.

  According to UN estimates, the world's population now exceeds 7.1 billion. Breeding of high-yielding varieties using wheat semi-fertility genes initiated by Dr. Borrogue “Green Revolution” (1940-60s) contributed greatly to increasing conventional grain production by a factor of 2.5 did. This increase in yield played a major role in feeding the rapidly growing global population at that time.

  However, since 1990, when the revolutionary green varieties were spread all over the world, the unit yield of cereals has not increased, and the food needed for the growing population has increased on cultivated land that has been opened up by expanding the rainforest. This is the current situation. Deforestation reduces the amount of photosynthesis, causing the global environment to deteriorate and biodiversity to decrease.

  On the other hand, so-called global warming, which raises the average temperature of the Earth's atmosphere and the ocean, has been taken up as a major problem, and the main factor is the increase in carbon dioxide emissions caused by the burning of fossil energy such as oil and coal. It is thought that there is. In order to reduce the amount of carbon dioxide released, conversion to renewable energy such as wind power, solar power generation, geothermal heat, and plant biomass is being considered. Since plant biomass is an organic substance in which carbon dioxide is fixed by photosynthesis using the energy of sunlight, even if the plant biomass is burned to extract energy, the amount of carbon dioxide released does not increase (carbon neutral). Therefore, it is expected as a means for simultaneously solving the two problems of securing energy resources and reducing carbon dioxide emission.

  However, the problem for putting renewable energy into practical use is poor in cost performance compared to fossil energy. In particular, the production of plant biomass competes with the production of food. There are concerns that it will lead to higher food prices.

  In agriculture in Japan, the production cost of crops that are too high is the biggest problem. For example, the production cost of 1 kg of rice is about 13,000 yen, which is almost the same as the sales amount. In addition, foreign sugarcane costs 4,000 yen / ton, while domestic sugarcane costs 20,000 yen / ton. Currently, the difference of 16,000 yen is covered by subsidies. In particular, Japan is currently considering participation in the Trans-Pacific Strategic Economic Partnership Agreement (TPP), and if TPP comes into effect, a large amount of foreign-produced cheap agricultural products will be imported, which will have a major impact on Japanese agriculture. There is no doubt.

  Also, against the backdrop of the rapidly increasing world population, there is a risk that the current food exporter may turn into a food importer, and there is no guarantee that Japan will continue to supply food in the future, making it easy for Japan to produce food. Relying on overseas is very dangerous from the viewpoint of food security. That is, the production of plant biomass in Japan is also effective for securing food production.

  In order to solve the various problems described above, it is effective to greatly increase the production amount of plant biomass per area. If the production cost of plant biomass can be reduced, industrialization of biomass energy can be promoted. In addition, it can greatly contribute to the profitability of agriculture and vegetable factories, and the practical use of material production using plants.

  By the way, as one of the methods for increasing the growth amount of eukaryotes such as plants, there is a method using hybrid stress. Hybrid stress is a genetic phenomenon in which hybrids between distant parents show stronger growth than parents, and has been established as a method for producing a primary hybrid (F1) variety.

  However, it is known that the increase in the amount of growth due to the hybrid strength is expressed only in the first generation of the hybrid and not inherited in the subsequent generation.

  On the other hand, with the development of gene analysis technology in recent years, the functions and gene networks of many genes in plants have been analyzed, and the data has been accumulated. Utilizing these research results, the biomass production of plants has been enhanced. There is a lot of research going on.

  For example, Eriksson et al. (2010) shows data indicating that the above-ground biomass of the poplar transformant in which the regulatory gene of the gibberellin biosynthesis system is highly expressed increases by about 70%.

  See also Ishimaru et al. (2012) reports that deletion of the IAA-glucose hydrolase gene expressed in rice seeds increases the grain weight and can increase the yield by 15%.

  Also, Kim et al. (2013) reported that the yield of a transformant of rice introduced with the OsDHAR gene, which increases the ability to process intracellular peroxides, was increased by about 15%.

  Recently, Yoshida et al. (2012) found a taw1-D gene that promotes the differentiation of secondary branch of rice panicle, and Koshihikari introduced with this gene by mating doubles the number of seed grains and increases the yield by about 50%. It is expected that it will become.

  Although each of the above reports is certainly effective, there has been no known research example that has increased the growth amount (dry matter weight) of the whole organism several times.

  Accordingly, an object of the present invention is to provide a method for producing a transformant that increases the growth amount (biomass) of a eukaryote such as a plant several times by gene transfer, and a transformant produced by the technique. And

A method for producing a transformant of a eukaryotic plant according to one aspect of the present invention that solves the above-described problem is obtained by using a promoter sequence of a ubiquitin gene transcribed by RNA polymerase II in rice isolated from different species . A transformation vector containing a construct ligated with 45S rRNA gene is used. Moreover, it is desirable that the plant is Arabidopsis.

Also, in a transformant of a plant that is a eukaryote according to another aspect of the present invention, a rice 45S rRNA gene isolated from a different species is linked to a promoter sequence of a ubiquitin gene transcribed by RNA polymerase II. It is produced using a transformation vector containing the constructed construct. The plant is preferably a transformant characterized in that it is Arabidopsis.

  A eukaryotic transformant according to another aspect of the present invention for solving the above-described problem is a transformant comprising a construct in which a 45S rRNA gene isolated from a different species is linked to a promoter sequence of a gene transcribed by RNA polymerase II. It is created using a conversion vector.

  As described above, according to the present invention, it is possible to provide a method for producing a transformant that increases the growth amount (biomass) of a eukaryote such as a plant several times by gene transfer, and a transformant produced by the technique. It becomes.

  In particular, according to the present invention, the biomass can be increased more than twice by producing a transformant that transcribes 45S rRNA gene with RNA polymerase II. It is different and original.

  Further, according to the present invention, it is possible to produce a transformant having a growth amount (biomass) increased several times without impairing the characteristics of various plant original lines (varieties). If the production amount per unit area can be increased several times, the production cost can be reduced to a fraction, so that the cost performance of biomass energy using sugarcane, eucalyptus, etc. can be improved. Moreover, if biomass can be increased, the production amount of grains, vegetables, and medicinal plants can be increased. In addition, it can contribute to the profitability of horticultural crop production such as plant factories. Furthermore, it becomes possible to increase the efficiency of substance production of useful substances using cultured cells such as plants. Plants such as orchids and apples take several years to blossom, but it can also be expected to shorten this maturity.

It is a figure explaining PCR amplification of 45S rRNA gene of rice, and construction of a binary vector. It is a schematic diagram about production of an Arabidopsis transformant. A rice 45S rRNA gene was ligated to a maize ubiquitin gene promoter and incorporated into a binary vector containing a hygromycin resistance gene to produce an Arabidopsis transformant by the Floral dip method. In this transformant, the rice 45S rRNA gene is transcribed by Pol II. It is a figure which shows the result of PCR and a Southern analysis of an Arabidopsis transformant. Two and three Arabidopsis transformants into which 4 and 3 copies of the rice 45S rRNA gene were introduced were produced. It is a figure which shows the time change of the growth test of the T2 progeny of a transformant 2 system | strain (45-2, 45-3), and a control (Col-0) system | strain, the number of expansion leaves, and the maximum diameter of a rosette leaf. Maximum diameter (DM) of rosette leaves at flowering stage of T2 progeny of two transformed lines (45S-2, 45S-3), dry matter weight (DW) of aboveground part. A value (D.W. ratio) where the dry weight of the control (Col-0) line was 100. FIG.

  Hereinafter, the form for implementing this invention is examined in detail. However, the present invention can be implemented in many different forms, and is not limited only to the embodiments and examples described below.

  In the method for producing a eukaryotic transformant according to this embodiment, at least a part of a 45S rRNA gene isolated from a different species is linked to a promoter sequence of a gene transcribed by RNA polymerase II (Pol II). A transformation vector containing the construct is used.

  In addition, the eukaryotic transformant according to the present embodiment includes a construct comprising a promoter sequence of a gene transcribed by RNA polymerase II and at least a part of a 45S rRNA gene isolated from a different species. Created using a vector.

  In this embodiment, the promoter sequence can be any promoter sequence as long as it is that of a gene transcribed by Pol II, but it must be a promoter sequence that can induce strong gene expression such as cauliflower mosaic virus. Is preferred.

  In this embodiment, the 45S rRNA gene is not limited, but it is preferable to use a 45S rRNA gene isolated from an organism of a genus different from the host organism into which the gene is introduced.

  Using a heterologous 45S rRNA gene linked to a promoter sequence and a terminator sequence recognized by Pol II as a target gene cassette, a transformant such as a plant into which the gene cassette has been introduced using a direct or indirect transformation method is produced. Can do.

  The transformation method is not limited, and any of a direct transformation method such as a gene gun (particle gun) method and an indirect transformation method such as an Agrobacterium-mediated method can be used. .

  Note that the method for producing a transformant exhibiting vigorous growth according to this embodiment can be applied to all plants such as dicotyledons, monocotyledonous angiosperms, gymnosperms, ferns, mosses, and algae. Hereinafter, the specific procedure will be described using a plant as an example, but in principle, it can be applied to a eukaryote other than a plant.

[45S rRNA gene isolation]
The method uses at least a portion of a plant 45S rRNA gene. The 45S rRNA gene can be amplified by PCR or excised from genomic DNA. It is also possible to use 45S rRNA genes isolated from eukaryotes other than plants.

[Method for constructing transformation vector]
The full length or a part of the isolated 45S rRNA gene is inserted between a promoter sequence and a terminator sequence of a gene transcribed by RNA polymerase II to prepare a fusion gene. Although it does not necessarily limit as a promoter arrangement | sequence, It is preferable to use a promoter arrangement | sequence with high gene expression level, such as a 35S transcript promoter of a cauliflower mosaic virus. Also, as described above, promoter sequences of eukaryotic organisms other than plants can be used.

  Then, the prepared fusion gene is incorporated into a vector containing a selection marker gene such as an antibiotic. In order to produce a transformed plant, it is preferably incorporated into a binary vector capable of replicating with Agrobacterium. However, it is also possible to produce a transformant using a direct gene transfer method.

  Moreover, in order to produce a transformed plant, it is preferable to use Agrobacterium (A. tumefaciens) holding Ti plasmid. In addition, it is also possible to produce a transformant using a perectal gun, microinjection, liposome, polyethylene glycol or the like.

  Although it does not necessarily limit as a transformation method using Agrobacterium, the binary vector method is preferable. The binary vector method is a method in which a plasmid in which a target foreign gene is incorporated into a T-DNA region of a plasmid having a border (LB and RB) of the T-DNA region is introduced into Agrobacterium to infect a plant. This is a method of inserting a gene into a plant genome.

  An expression cassette using the binary vector method includes, in the T-DNA region, a 45S rRNA gene of a target gene, a selection marker gene for selecting a cell into which the gene has been introduced, and a reporter gene. A marker-free (MAT) vector can also be used.

  Examples of promoter sequences include cauliflower mosaic virus 35S RNA, nopaline synthase (nos), actin, and β-phaseolin, napin, and ubiquitin promoters that induce strong gene expression in plants. Also, eukaryotic promoter sequences other than plants can be used.

  The terminator sequence may be any sequence that can terminate the transcription of the gene transcribed by the promoter. Examples thereof include octobin synthase (ocs), cauliflower mosaic virus 35S RNA gene terminator, and nopaline synthase ( nos) gene terminators are preferred.

  As the selection marker gene, a gene that imparts resistance to drugs such as antibiotics and herbicides is used. Specific examples include a kanamycin resistance gene, a paromomycin B resistance gene, or a resistance gene for herbicides such as bispyribac sodium salt (BS), bialaphos, glufosinate, and glyphosate. It is also possible to use a marker-free vector such as the MAT vector system.

  In the present embodiment, an enhancer sequence or the like may be further included as necessary. The enhancer is used to increase the expression efficiency of the target gene, and is not limited, but examples include an enhancer region including an upstream sequence within the promoter of the 35S RNA gene of cauliflower mosaic virus. be able to.

  The binary vector contains the above expression cassette in the T-DNA region, and specifically, a pEKH or pEKB vector in which the above expression cassette is incorporated is preferred, but pBI, pRI, pPZP A vector in which the above expression cassette is incorporated into a commercially available vector such as a serotype, pSMA type, or pGW type can also be used.

  Then, the Agrobacterium for gene introduction is introduced by introducing the binary vector having the above-described configuration into Agrobacterium by a triple parental mating method, a free-thaw method, an electroporation method, or the like. Can be prepared.

[Preparation of Agrobacterium inoculum]
As an Agrobacterium inoculum for gene introduction, for example, a culture solution obtained by culturing Agrobacterium holding a foreign gene in 2 × YT medium, AB medium, YEP medium, LB medium or the like for about 15 to 24 hours is used. Alternatively, it may be prepared by collecting cultured Agrobacterium and suspending it in an MS medium containing MES and acetosyringone, and using the culture solution as it is as an inoculum. May be.

  The inoculum is not limited, but preferably contains a surfactant. Surfactants reduce the surface tension of water, thereby allowing the Agrobacterium suspension to penetrate into the plant tissue.

  The surfactant used may be any surfactant that can reduce the surface tension of water and promote the penetration of the inoculum into the plant tissue. For example, SILWET L-, which is commercially available as a transformation reagent. 77, Tween 80, Tween 20, Triton X-100, and the like. Moreover, although the density | concentration of surfactant is based also on the kind of surfactant, 0.005-0.02% aqueous solution is used preferably.

(Preparation of explants)
Plant tissues subjected to gene transfer include leaf pieces, hypocotyls, and callus. These tissues are preferably prepared from plants maintained aseptically. However, it is also possible to prepare directly from a plant grown in a greenhouse or the like by sterilization treatment. In addition to the above, cultured cells can be used when a transformant is produced using the direct gene transfer method.

[Gene transfer]
An explant is prepared as described above, and the target gene held by Agrobacterium is introduced into the plant cell by immersing it in an Agrobacterium inoculation solution holding the target gene. In the direct gene introduction method, a DNA fragment containing a target gene is physically introduced into a target eukaryotic cell.

  Cells into which the 45S rRNA gene has been introduced show vigorous growth from that time. However, it is preferable to breed progeny lines and select lines that have the 45S rRNA gene homologously. This makes it possible to produce a transformant in which stress growth is genetically fixed.

  The 45S rRNA gene used in this method is preferably isolated from a species of a taxon different from the target plant (host). This does not deny that the growth is induced by 45S rRNA isolated from the same species, but since it is not possible to distinguish 45S rRNA transcribed by RNA polymerases I and II, It is difficult to prove the causal relationship with growth.

  As described above, according to the present embodiment, a method for producing a transformant that increases the growth amount (biomass) of a eukaryote such as a plant several times by gene introduction and a transformant produced by the technique are provided. It becomes possible. Moreover, since the increase in the amount of growth according to the present invention is inherited to progenies, genetically fixed varieties can be produced.

  In particular, according to the present invention, the biomass can be increased more than twice by producing a transformant that transcribes 45S rRNA gene with RNA polymerase II. It is different and original.

  In addition, according to the present embodiment, it is possible to produce a transformant in which the growth amount (biomass) is increased several times without impairing the characteristics of various plant original lines (variety). If the production amount per unit area can be increased several times, the production cost can be reduced to a fraction, so that the cost performance of biomass energy using sugarcane, eucalyptus, etc. can be improved. Moreover, if biomass can be increased, the production amount of grains, vegetables, and medicinal plants can be increased. In addition, it can contribute to the profitability of horticultural crop production such as plant factories. Furthermore, it becomes possible to increase the efficiency of substance production of useful substances using cultured cells such as plants. Plants such as orchids and apples take several years to blossom, but it can also be expected to shorten this maturity.

  Hereinafter, the above method was actually practiced to confirm the effect of the present invention. This will be specifically described below. The following examples are merely examples and do not limit the scope of the present invention.

[Isolation of 45S rRNA gene]
First, genomic DNA was extracted from the leaves of an Indian type strain N16 of a rice cultivar (Oryza sativa L.) by the CTAB method (Doyle and Doyle 1987).

  Subsequently, the full length (5.8 kb) of the 45S rRNA gene was amplified by PCR, and the 45S rRNA gene was PCR amplified. The following amplification primers were used. Then, after 94 ° C for 3 minutes, the cycle of 94 ° C for 1 minute, 58 ° C, 1 minute, and 68 ° C for 5 minutes was repeated 30 times. Cloned (see FIG. 1).

  Then, EcoRI sites at both ends of the full length of the isolated rice 45S rRNA gene were subjected to partial digestion and agarose electrophoresis to gel extract a fragment containing the rice 45S rRNA gene. Thereafter, the extracted fragment is blunt-ended, the BamHI site and SalI site of the Sub221s vector are cleaved, blunt-ended, and the promoter sequence of the maize ubiquitin gene and the terminator sequence of the nopaline synthase gene of Agrobacterium A fusion gene (UbiP :: 45S rRNA) was prepared by inserting in between (see FIG. 1).

  Then, a plasmid containing a fusion gene similarly cleaved with HindIII is inserted into the HindIII site between the kanamycin and haogromycin resistance gene cassettes held by the binary vector (pEKH2) to construct pEKH2 UbiP :: 45S rRNA. did. Since the Sub221s vector contains a spectinomycin resistance gene, a binary vector containing the target gene could be efficiently selected (see FIG. 1).

  And pEKH2-UbiP :: 45S rRNA was introduce | transduced into Agrobacterium (Agrobacterium tumefaciens) strain EHA101 by Triparal mating method using colon_bacillus | E._coli containing the constructed binary vector pEKH2 UbiP :: 45S rRNA.

  Then, 2 × YT medium not containing antibiotics containing spectinamycin 50 mg / l, kanamycin 25 mg / l and chloramphenicol 25 mg / l was inoculated with Agrobacterium containing rice 45S rRNA gene and cultured for 16 hours. did. The bacterial solution was centrifuged at 4,000 rpm, 25 ° C. for 3 minutes. Thereafter, the supernatant was discarded, and after resuspension in 1 / 2MS medium supplemented with 5% sucrose and MES, L-77 was added to 0.02% and stirred well. The Agrobacterium concentration (OD600) was adjusted to about 1.0.

[Method for producing transformed plant]
The seeds of the model plant Arabidopsis thaliana Columbia-0 were aseptically sown in 1/2 MS medium containing 3% sucrose, and the seedlings 2 weeks after germination were potted, 22 ° C., 16 hours, light period, 8 hours, dark period Cultivated in a controlled growth chamber. After potting, cocoons for about 3 weeks were used for Agrobacterium inoculation.

  Then, 10 to 20 μl of Agrobacterium suspension was dropped onto the tip of the inflorescence of Arabidopsis 35 days after seeding using a pipetman. About 30 days after inoculation with Agrobacterium, mature Arabidopsis seeds (T1 generation) were successfully cultivated.

  In the Arabidopsis transformant produced by this method, it is considered that the Arabidopsis 45S rRNA gene is transcribed by RNA polymerase I, and the rice 45S rRNA gene linked to the promoter sequence of the introduced corn ubiquitin gene is transcribed by RNA polymerase II ( (See FIG. 2).

[Selection of transformed seeds]
The dormancy of the seeds obtained above was broken by storing in a refrigerator overnight, and aseptic seeding was carried out in 1/2 MS medium containing 3% sucrose supplemented with hygromycin at a concentration of 20 mg / l.

  Then, after 20 days, 5 individuals resistant to hygromycin were selected and transplanted to a pot filled with No. 2 pot with soil mixed with Metromix and roof soil in a 1: 1 ratio.

  Leaves were sampled from individuals that had passed 10 days after potting, and DNA was extracted by the CTAB method. Using the extracted DNA as a template, PCR was performed using primers that amplify the hygromycin (hyg) gene. PCR conditions were 94 ° C for 3 minutes, and then a cycle of 94 ° C for 1 minute, 59 ° C, 1 minute, 68 ° C for 1 minute was repeated 30 times. When the PCR amplification product was subjected to agarose gel electrophoresis, the hyg gene could be detected in all 5 individuals (see FIG. 3).

  Then, genomic DNA was cleaved with restriction enzymes for 5 lines in which amplification of the selection marker (hyg) gene fragment introduced together with the 45S rRNA gene of rice was confirmed by PCR, subjected to agarose electrophoresis, and Southern. When the copy number of the introduced hyg gene was analyzed, 4 copies were found in 2 lines (45S-1, 45S-2), and 3 copies in the remaining 3 lines (45S-3, 45S-4, 45S-5). The hyg gene was introduced (see FIG. 3). The Southern blotting method was carried out in accordance with the Roche DIG application manual.

  Of the obtained 5 lines of transformants, 3 lines were poorly grown, but the remaining 2 lines (45S-2 and 45S-3) grew well, so they were self-bred to generate the T2 generation. And T3 generation ripe seeds.

  Furthermore, the dormancy of T3 generation seeds was overcome by storing in a refrigerator overnight, and aseptically sown in 1/2 MS medium containing 20 mg / l hygromycin. The Col-0 line used for the control was similarly performed under the condition not containing hygromycin.

  Then, 15 days after aseptic seeding, 8 strains of 2 transformants and 8 control Col-0 strains were transplanted into pots filled with No. 2 pot with soil mixed with Metromix and roof soil one-on-one.

  Then, the number of the developed true leaves of the two transplanted transformants (45S-2, 45S-3) and the control strain (Col-0) was counted until the 25th day after sowing. Further, the maximum diameter of rosette leaves was measured every 2 days up to the 40th day after sowing (see FIG. 4).

  Then, on the 40th day after sowing, the maximum diameter of the rosette leaf of the plant that reached the flowering stage was measured. Further, the entire above-ground part was harvested and dried at 120 ° C. for 1 hour and 65 ° C. for 12 hours, and the dry weight was measured using a precision balance.

  As a result, the two lines (45S-2 and 45S-3) introduced with the rice 45S rRNA gene were compared with the original line Col-0 in the maximum diameter of the rosette leaves and the above-ground dry weight 40 days after sowing. The value was significantly large. In particular, the dry weight of the 45S-2 line was 2.8 times that of the control (see FIG. 5).

  As described above, it was confirmed that a plant body showing vigorous growth can be created by creating a transformant in which a fusion gene in which a heterologous 45S rRNA is linked to a promoter sequence transcribed by RNA polymerase II of this method is introduced. . Since this technique follows the basic principles of eukaryotes, it can provide transformants that show vigorous growth in all plant species and non-plant eukaryotic species.

The present invention has industrial applicability as a transformant and a production method thereof.

Claims (2)

A transformant of a eukaryotic plant transformant using a transformation vector comprising a construct in which the promoter sequence of a ubiquitin gene transcribed by RNA polymerase II is linked to the full length of rice 45S rRNA gene isolated from a different species. In the production method , a method for producing a transformant, wherein the plant is Arabidopsis . A plant that is a eukaryote produced using a transformation vector comprising a construct in which the promoter sequence of a ubiquitin gene transcribed by RNA polymerase II is linked to the full length of a rice 45S rRNA gene isolated from a different species. A transformant , wherein the plant is Arabidopsis .